Calcium/phosphatidylserine(PS)-dependent protein kinase C (PKC),an enzyme that phosphorylates numerous cellular proteins, is activated by phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DG). Recently we have shown that PIP2, like DG, increases the affinity of PKC for calcium, translocates it from soluble to particulate fraction, displaces phorbol esters from PKC and induces the autophosphorylation of PKC. However, the physiological significance of PIP2.PKC is not yet known. The principal objective of this proposal is to elucidate the functional role of PIP2 in PKC-mediated protein phosphorylation, and to study interactions of other cellular components in this process. Preliminary studies have indicated that both 4- and 5 phosphate groups of PIP2 are essential for the activation of PKC, and that the activity of the enzyme is dependent on the pH. Alkalinization of the medium was found to increase the Vmax of PIP2.PKC. Na+/H+exchanger-mediated alkalinization of the cytoplasm is a common response to mitogens. The possibility will be explored if DG.PKC and PIP2.PKC phosphorylate different regions of the Na+/H+ exchanger (a PKC substrate) resulting in turning on and off of the exchanger. The two important steps in PKC activation, i.e. autophosphorylation and translocation of PKC from soluble to membrane fraction which were found to be influenced by PIP2, will be studied as a function of pH. These studies may help in understanding the role of alkalinization which always precedes signal transduction. Other studies will focus on the possible role of spermine, an intracellular polycation, in PIP2.PKC-mediated phosphorylation process. The structure -activity relationship of PIP2 will be investigated by modifying different regions of PIP2, and studying the effect of these alterations on PKC-activity. Studies will include effects of a) hydrogenation of fatty acyl chains of PIP2, b) Lyso-PIP2, c) acetylation of inositol (partial or complete), d) ketalization (necessity of 2- and 3-OH) and e) 2-3 splitting (is the inositol ring required?) on PKC activation. Further studies will be conducted on the effect of phosphatidylinositol 3,4,5-trisphosphate (PIP3, a recently discovered phosphoinositide formed by phosphorylation of PIP2) on PKC activity. It is anticipated that PIP3 may activate PKC more efficiently than PIP2 because the activation of PKC has been found to depend on phosphorylation status of phosphoinositides. G-protein (Gs PIPK) is involved in the stimulation of PIP-kinase which results in the production of PIP2. The mechanism of receptor-coupled activation of PKC by PIP2 will be studied by reconstituting Gs PIPK/PIP-kinase systems, and studying its effect on PKC-stimulation.